Project Summary Enzymes are proteins that aid in the acceleration of metabolism or the chemical reactions in all living organisms. By synthesizing certain molecules and degrading others, enzymes can catalyze a range of biochemical reactions both in vivo and in vitro. When in collaboration with transporters and receptors, enzymes regulate almost all physiological functions in the body. Therefore, it is important to thoroughly understand ex- and in vivo enzyme activities. Among the many methods of studying enzyme activities, time-resolved macromolecular crystallography (TRX) has the advantage of investigating enzymatic reaction details on the fly. However, TRX theoretically can only capture the states where the enzymes are at local energetic saddle points. On the other hand, modern hybrid quantum mechanics/molecular mechanics (QM/MM) enables studying enzymatic reactions where new molecules are formed or destroyed. However, without the support from solid biological structures or if the transformation between reactant and product states is distinctively different, QM/MM cannot reach the authentic answer. This proposal aims to establish a new TRX- and QM/MM-based strategy to investigate enzymatic activities by joining the strength of those two territories. Notably, a recently elucidated allosteric controlling mechanism in 70- kDa heat shock proteins (Hsp70s) leads to an unparalleled opportunity of building a model system as a benchmark for developing the proposed TRX-QM/MM strategy. Specifically, in Aim 1, a groundbreaking discovery of oxygen radicals driving ATP hydrolysis will be examined by TRX experiments on the ATPase domain of bacterial Hsp70 DnaK. In Aim 2, I will use QM/MM to identify and verify the oxygen radical species, and depict the free energy landscape of the hydrolysis event in full scale. In Aim 3, I will use DnaK and actin to benchmark the development of three components that will significantly enhance the scope of the TRX-QM/MM method, including an automated freezing-and-quenching instrument, a new electron diffraction method for chasing proton transportation, and a new crystallographic refinement program that can handle open-shell systems. During the K99 phase (Aim 1 and 2), I will be mentored by Dr. Wayne Hendrickson, a leader in macromolecular crystallography, and Dr. Arieh Warshel, a leader in computational enzymology. This work will reveal a novel mechanism of ATP hydrolysis by Hsp70 in the short term. Still, most importantly, it will establish an unparalleled TRX-QM/MM method for broad enzymatic studies in the longer term.

Project Narrative Enzymes can indispensably catalyze biochemical reactions both in vivo and in vitro, and thus deserves a thorough understanding of their reaction details. There is a lack of a method to study enzymatic reaction details offering both ultimate scale and accuracy. This proposal aims to establish such a method by jointly employing time-resolved crystallography and computational enzymology, and by developing a set of accompanied new tools based on benchmarking with the 70-kDa heat shock protein (Hsp70).